SE415036B - COMPOSITION OF ENZYMATIC SUBSTRATE AND CARRIER AND ITS USE IN AN INDICATOR DEVICE - Google Patents

Description

7405199-6 in Table 1 several references, relating to carrier-bonded substrates, and for these references is that they refer to substrates, which are chemical bound to a support, i.e. by covalent bonding or ionic bonding. (anionic). The intention is to use the carrier-bound substrates For the pure production of enzyme L. In this case, only one enzyme -substrate complex formation without degradation of snbstrat. According to it U.S. Pat. No. 2,677,917 and French Pat. No. 2,071,058, it is further known that on carriers, such as charcoal or expanded perlite, apply nutrient medium for fungal mycelium lium. The nutrient media used are water soluble and enabling for the fungal mycelium to absorb the nutrient medium in a moist medium. to support growth. The corresponding is described in it U.S. Patent 3,251,749 to bacterial conversion to polysaccharides of carbohydrates, which are applied to carriers. Also in this case the "substrate" is water soluble and only loosely brought on the wearer.

As further examples of the prior art can be mentioned French Patent Specification 1,306,640, which describes containment of enzyme or enzymatic substrate in a permeable material, as a polymeric material. French patent specification 595 782 writes application of sulfuric acid on diatomaceous earth. The British Exhibit 891 467 describes the application of an active substance, capable of oxidizing or reducing (quinone- hydroquinone), on various carriers. The U.S. Patent 2,717,852 discloses the application of enzyme to carriers. The French U.S. Patent No. 940,108 relates to reinforcing fillers for rubber, which filler consists of fine-grained mineral material coated with a surfactant or lubricant, such as oil or grease. U.S. Patent 1,979,380 describes the coating of pigment particles, such as carbon black, with organic material, such as linseed oil. The British patent 993 944 describes the coating of sodium aluminosilicate with large surface area with a dye. The U.S. Patent 2,354,318 discloses coating of fine-grained calcium carbonate with an adhesive, the product being intended as a pigment pelvis in wallpaper. IN Contrary to what is thus known, the present invention to a particular type of carrier-bonded substrate, wherein the substrate is not covalently bonded to the carrier, either directly or via couplers, but by interaction between the substrate 3 7405199-6 and a special carrier with a large specific surface area is firmly bonded to the carrier by adsorption. It is also a condition of invention that the substrate has low solubility in water and does not sink hydrolysis or dissolution in the absence of the corresponding enzyme.

* In contrast to some of the above mentioned patents, in which the coating consists of loosely applied, water-soluble material, is the substrate thus solidly adsorbed in the present invention to the carrier and also sparingly soluble in water. Thereby obtaining read according to the invention in a very simple and inexpensive manner one to a carrier fixedly bound enzymatic substrate. For the influence of the carrier-bonded substrate according to the invention, a single zym in the aqueous phase and through the nature and properties of the carrier bonded substrate according to the invention a high and controllable is achieved affinity between the enzyme and the substrate.

According to the invention there is thus provided a composition of enzymatic substrate and carrier, which composition is characterized that, in addition to any additives in the form of diluents, and / or viscosity modifiers, consists of l) a solid, inert to the enzymatic substrate carriers with a specific surface area of at least 50 m2 / g, and 2) a layer bonded to the support by adsorption, which contains a water-soluble enzymatic substrate, which has ability to undergo enzymatic reaction in the presence of the equivalent enzyme, but in its absence is stable and free from hydrolysis and resolution. .

The diluent is preferably selected from mineral oils with several hydrocarbons, higher aliphatic alcohols and polyglycols.

The viscosity altering agent may be metal soaps, for example aluminum stearate. ' IN According to a further preferred embodiment of the invention the carrier has a specific surface area of at least 200 m2 / g. 7 The carrier is preferably selected from the organic / inorganic polymer. meres, inorganic oxides, acids and salts.

It is especially preferred to choose the support from alumina, calcium carbonate, magnesium carbonate, silica aerogel and calcium carbonate phosphate.

According to a particular embodiment of the invention, the substrate is glycerin tricapronate.

A particular aspect of the invention relates to an enzymatic indication vuos199-6 4 which comprises a carrier-bound composition of enzymatic substrate In combination with an enzyme corresponding to the substrate and an indicator indicator for the reaction formed during the enzymatic reaction tion product.

The solid bond (adsorption) that exists between the substrate and the carrier in accordance with the present invention, by using a special carrier, which, as mentioned above, is solid and inert to the enzymatic substrate and having one specific surface area of at least 50 m2 / g, preferably at least 200 m2 / g. Through the resulting solid bond avoids any unwanted separation of the substrate from the carrier. Although it is not entirely clear, we should the markedly increased affinity which occurs between the carrier the bonded substrate of the present invention and the corresponding zym, can be attributed to the combination with the particular carrier and then mainly its large specific surface area. It has in the invention found that with increasing specific surface area of the carrier, a increasing activity or reaction rate between substrate and enzyme. Thereby, an activity increase of up to 10 times has the normal activity obtained.

Without any comprehensive enumeration of all those against different enzymes corresponding substrates, which are known, it will be appreciated that the invention is applicable to any substrate which meets the above requirements ie the substrate must be sparingly soluble or substantially insoluble in water, by which is meant that the substrate should have a solubility of more than 0.1% by weight in water at 20 ° C, and the substrate should further be stable and not undergo hydrolysis or dissolution in the absence of the corresponding enzyme. Preferred substrates in the present invention invention are those selected from glycerin tricapronate (tricaproin), tripelargonine, tributyrin and bis-3,5,5-trimethylhexyladipate, mixed esters between polyhydric alcohols and organic and inorganic ror.

The carrier material, which is presently particularly preferred, is silica in the form of an aorogel with a specific surface area of about 200-300 m2 / g. Such silica aerogel is commercially available under the names "Aerosil", "Cab ~ O-Sil", "silica gel G" from the company Merck and "Fluosi1" (Nynäs Petroleum AB, Sweden).

The carrier-bonded substrate of the present invention is preferably prepared by applying the substrate to the carrier by impregnation, the finished product being in the form of a easy-to-handle, storage-stable powder. which is free-flowing. Thus 7495199-6 the carrier is suspended in a liquid containing substrate or substrates and substrate diluents with stirring. After this treatment evaporates the added liquid. The residue is comminuted so that particles of desired size and size distribution are obtained.

In its simplest and most basic embodiment includes the invention carrier material with a layer of a substrate of the above defined kind. As previously stated, the carrier should be solid and chemically inert to the substrate and having one surface area per unit weight of at least 50 m2 / g.

According to another embodiment of the invention, it is constituted on the carrier material did not apply the layer of substrate alone but of substrate in combination with a diluent. In the layer on the carrier material, the substrate will thus be dissolved in the diluent. In contrast to the previously described embodiment the form, where the whole substrate is directly accessible for the action of enzyme, in this latter embodiment is essentially only that substrate material present in the outer part of the layer directly available for the action of enzyme. The diluent's own such as solubility of substrates, activating or inhibiting enzyme effects, viscosity and propensity for separation during effect of low temperature, affects the availability of the substrate for influence of enzyme. Suitable diluents are mineral oils, higher aliphatic alcohols such as decanol, dodecanol, stearyl alcohol and polyglycols and other volatile hydrocarbon compounds, which are miscible with the substrate.

A further influence on the availability of the substrate for effect of enzyme is obtained by adding viscosity-changing part, such as aluminum stearate, to the diluent.

The substrate composition of the present invention is reversible for any suitable purpose for reaction under the influence of associated enzyme. It is extremely advantageous to use the take carrier-bound substrates in standardization and characterization of enzymes. A particular field of technology, within which the present invention has proved to be particularly useful, indicators of -temperature-dependent indication, eg of food and then also refrigerated and frozen foods. Such indicators are known by the front all U.S. Patent 2,671,028.

Thus, if an indicator comprising an enzyme and its sub- and an indicator substance for indicating it at the enzyme level. the reaction product formed, is subjected to the same 7465199-6 storage conditions as a food, it is possible that, where appropriate, calibration of the indicator, by the color change of the indicator determine food storage conditions.

A corresponding enzymatic indicator, which uses the carrier the bonded substrate of the present invention, comprises as active components said carrier-bonded substrates in combination with an enzyme adapted to the substrate and an indicating substance, for example a pH indicator, for indicating the enzymatic reaction reaction products formed.

When using the carrier-bonded substrate according to the invention and the corresponding enzyme in aqueous suspensions, can thickeners, such as water-soluble, high molecular weight compounds compounds, such as agar or carboxyellulose, are added to the suspension. nen. In this case, an advantage is obtained by the thickener, e.g. agar, gives a certain change in the reaction rate.

To further clarify the invention, it comes below to be described by way of example. It should be noted, however, that the examples given are illustrative only and not limiting. It should It is further emphasized that in addition to the exemplified enzymatic systems but of course many other enzymatic systems are useful and understood within the scope of the invention. åšemgel l Preparation of substrate coated carrier.

In 2500 ml of methylene chloride was immersed in small portions and under stirring 100 g of fine-grained silica ("Fluosil" from Nynäs Petro- leum, Sweden) until a homogeneous slurry was obtained.

The slurry was then mixed with 100 g of glycerol tricapronate (tricaproin, TC) and stirring was continued for another hour at room temperature.

Thereafter, the methylene chloride was removed by air blowing through filter nozzles and slight heating to 30 ° C. When mixed the volume of the mixture was reduced to 1500 ml, the heating was stopped for to avoid shock boiling and the further evaporation of methylene chloride was done with air lock only during continuous stirring.

Final evaporation of the methylene chloride was performed in an oven at 0 ° C. After drying in the heating cabinet, a white, brittle mass, which was mortarized and sieved through a Sift mêå Ö flfi kad mesh size to obtain a fine-grained powder. The corresponding result was obtained when the dried, white, brittle mass was suspended in water. and ground in a colloid mill. v 7lr05199-'6 gxemgel 2 Preparation of carrier coated with substrate and diluent.

Fine-grained silica was slurried in methylene chloride on the same as in Example 1. A mixed mixture was then added to the slurry. 50 g of tricaproin and 50 g of spider oil with stirring and stirring was continued for 1 hour at room temperature.

In a manner similar to Example 1, the solution was then removed. the pulp and the resulting mass were ground in mortar and colloidal grinder. A fine-grained powder of carrier coated with substrate and diluent was obtained.

Egemgel 3 Example 2 was repeated with the difference that aluminum stearate in an amount of 10% was added to the spindle oil. Corresponding results as in Example 2 were obtained. läeamäsëli Example 2 was repeated but exchanging the spindle oil for stea ~ ryl alcohol. In this case, heating was required in order for the mixture to evaporate tricaproin and stearyl alcohol would become homogeneous. In other respects rapids as in Example 2 and corresponding results were obtained.

Example 5 Experiments were performed with the substrate preparations prepared in Examples 1-3. laid carriers. In the experiments, the time-temperature dependent the indicating properties when indicated in combination with the enzyme reaslipas. The acid formed in the enzymatic reaction indicates with a pH indicator. The weight ratio between carrier and it the substrate-containing layer on the support was in all cases 1: 1.

The results are given in Table 1, using the following abbreviations fi des in the table: Fluosil = F; trikaproin = TC; spindle oil = S; aluminum stearate = A1st. ßa.1_ ~ «.L _ .. f-v _. <> mee fi§_ tl§ Temp TC / F TC / S / F TC / S / l0% Alst / F <° c) +20 * 1.0 1.0 1.0 +4 2.4 2.0 1.7 -5 15.5 20.0 33 «L0 40.0 108 417 ~ l5 321 856 970 -20 l396 - * 740519996 The experimental results confirm what has been said before, namely that based on a certain time-temperature dependence at a with one bare substrate coated carrier increases this dependence when the substrate combined with diluents, ie the relative turnaround times forß along. Dependence is further increased to the combination of substrates diluents are added viscosity enhancing agents, such as aluminum stearate.

Example 6 To study the effect of thickeners in the aquatic containing suspension of enzyme substrate, 0.25% of agar was added to the water. containing suspensions of enzyme (pancreatic lipase) and substrate laid carrier according to example l. The obtained indication times note ~ and are given as relative turnaround times in Table 2.

Table 2 Relative turnaround time Temp TC / F 'TC / F (° C) 0.25% agar - +20 1.0 1.0 +4 2.4 2.4 -S 12.7 15.5 -l0 84.2 40.0 -15 1336 321 -20> l6000 1396 During the experiment, the effect of the agar at tempe ~ could be clearly felt temperatures below ~ 10 ° C. ßxemgel 7 Attempts were made to compare the activity under the influence of enzyme (pancreatic slipase) on substrate (tricaproin), partly without carrier, partly coated on carrier 1 in accordance with example l. In the experiment nn ¥ varying concentrations of enzyme and as a measure of activity indicated at 20 ° C formed reaction product by means of pH kator. The results are shown in Table 3.

Qabell Q 0mslagstid_in minutes Lipase concentration TC 'TC / F (ng / ml) 3 1 7.5 <1 l _ 15 1.3 0.3 f i 37 4.5 0.1 f 5 _ 140 11.5 0.03 1 '380' 32 9 1405199-6 Table 3 shows that the combination of substrate and carrier with a large specific surface area entails an approximately tenfold increase in compared to the activity of substrate alone. ßxemgel 8 As in Example 7, attempts were made to compare the activity by the action of enzymes on substrates, partly without carrier, partly coated on carriers. The enzyme used was pancreatic lipase, the substrate was of triple argon (TP) and the carrier was "Fluosil" V 300, which had one specific surface area of approx. 300 m2 / g. As a measure of activity was indicated reaction product formed at 20 ° C. The results are given in Table 4. ïabell 4 Wrapping time in hours (h) Relative enzyme concentration TP TP / F 32 _ 21 4.25 16 28 12 8 77 25 4 240 46 2 524 100 1 - 240 ëggngel 9 Example 8 was repeated, but with the difference that tripropionin (TPP) was used as a substrate instead of tripelargonine. The enzyme was pancreatic slipase and the carrier "Fluosil". The results are given in table 5. íiëseelii ' Wrapping time in hours (h) Relative enzyme concentration TPP TPP / F 32 l, 25 0.5 16 4.75 1.25 8 Zl 2.5 4 28 <2l 2 70 21 l 142 25 §§§MQel 10 Example 8 was repeated, but with the difference that bis ~ 3,5,5- trimethylhexyl adipate (THA) was used as the substrate instead of geranium. The enzyme was, as before, pancreatic slipase and the carrier "Flnosil". The results are given in Table 6. 7405199-6 10 ' Iëë§ll_š _ Wrapping time in hours (h) Relative enzyme concentration_ TKA THA / F 16 '70 21 8 384 35 4 - 71 2 ~ 192 l - 240 ~ 284 Examples 8-10 show that the combination of snbstrat and carriers with a large specific surface area lead to a sharp increase in compared to the activity when using substrates without a carrier.

Eggmpel ll It was used essentially according to Example 7, with that difference that instead of "Fluosil" they used "Kiselgel G" from the company Merck as a carrier. This carrier has the composition: 13% CaSO 4, 0.02% chloride, 0.03% Fe and the residue SiO2. The carrier grains have a diameter ter of 5-25 um. For the non-carrier substrate, one was used 1% solution of tricaproin in aeetone, while for the vehicle-bound sub- a suspension containing the corresponding amount was used silica gel coated with 30% tricaproin. After addition of enzyme (pank ~ reaslipase), pH 5.4 was noted at 28 minutes for the vehicle the substrate at 220 minutes for the non-carrier substrate at 2 ° C.

It is thus clear that by choosing appropriate combinations nations of substrates, substrate-diluents, substrate-diluents agent-viscosity modifiers, can create time-temporal indicators. temperature-dependent indication, which indicators are adapted for the conditions applicable to the storage of a particular good. It is also realized that by the manageable shape of the substrate according to the invention can further adjust the indicator by mixing the substrate materials of various kinds, ie mix materials that are only coated with substrates and materials coated with a kawbinntion of sub- strat ~ diluent-viscosity-changing agent, etc. The possible ~ opened to adjust or control the temperature dependence of the indicator are unforeseen and by using_the substrate according to the invention can be created indicators, which are adapted to each special purpose, it may be for the indication of photographic: ma ~ terinl, medicines, foods or other sensitive heat, as for its shelf life requires certain storage conditions.

Claims (9)

It should be emphasized that the above description is intended to be illustrative only and not restrictive. Obviously, many modifications and variations can be made which fall within the scope of the invention as defined in the appended claims. PATENT REQUIREMENTS Composition of enzymatic substrate and carrier, characterized in that, in addition to any additives in the form of diluents and / or viscosity-changing agents, it consists of 1) a solid, inert to the enzymatic substrate carrier having a specific surface area of at least 50 m2 / g, and 2) a layer bonded to the support by adsorption, which contains a water-soluble enzymatic substrate, which is capable of undergoing enzymatic reaction in the presence of the corresponding enzyme, but in its absence is stable and free from hydrolysis and dissolution . Carrier-bound composition according to claim 1, characterized in that the diluent is selected from mineral oils, higher aliphatic alcohols, ketones, ethers and polyglycols. A carrier-bound composition according to claim 2, characterized in that the diluent is spider oil, decanol, dodecanol or stearyl alcohol. . '4. Carrier-bound composition according to claim 1, characterized in that the viscosity-changing agent is aluminum stearate, agar or carboxycellulose. Carrier-bound composition according to any one of the preceding claims, characterized in that the carrier has a specific surface area of at least 200 m2 / g, preferably 200-300 m2 / g. Carrier-bound composition according to any one of the preceding claims, characterized in that the carrier is selected from organic / inorganic polymers, inorganic oxides, acids and salts. A carrier-bound composition according to claim 6, characterized in that the carrier is selected from alumina, calcium carbonate, magnesium carbonate, silica aerogel and calcium phosphate. 7405199-6 12 Carrier-bound composition according to any one of the preceding claims, characterized in that the substrate is glycerol tricapronate, tripelargonine, tributyrin, bis-3,5,5-trimethylhexyl adipate, tripropionine or mixed esters of polyhydric alcohols and organic and inorganic acids. . Use of the composition according to any one of claims 1-8 in an enzymatic indicator device. REFER TO PUBLICATIONS; us a zos 918 (195-66)